Diabetes mellitus is a disease characterized by marked changes in amino acid, lipid, and carbohydrate metabolism. In general, the metabolic status of the diabetic is catabolic in nature and, with regard to protein metabolism, a general wasting of tissue and proteolysis is observed. This increased production of amino acids in the peripheral tissues results in an imbalance in whole body nitrogen metabolism because of increased production of ammonia. Glutamine represents the main shuttle mechanism for transfer of toxic ammonia to the liver where the excess nitrogen is converted to urea and excreted from the body. Glutamine represents over 50 percent of the total free amino acid pool in the body and it is the amino acid found at the highest concentrations in both plasma and dependent active transport mechanisms. For example, the free concentration in liver is approximately 8 mM and in muscle it can be 20 mM despite a plasma concentrations of only 0.6 mM. The transport activity responsible for maintenance of these gradients in liver and muscle has been termed System N, a goal of the proposed research is to identify the System N carrier protein, produce antibodies against this protein, and clone a cDNA for the corresponding gene. These antibodies and cDNA probes will be utilized to investigate the regulation of the hepatic System N carrier in catabolic diseases such as diabetes in which glutamine flows between tissues become critically important to whole body nitrogen metabolism. Although it has been nearly 30 years since the first amino acid transport activity was characterized at the cellular level, none of the proteins responsible for the dozen or so known activities have been identified. The investigators intend to use a variety of approaches to identify and characterize the System N carrier including purification using a reconstitution assay, production of monoclonal and monospecific polyclonal antibodies, and identification of the System N mRNA through translation in microinjected Xenopus oocytes. The investigators have made significant progress in laying the groundwork for the proposed experiments and the likelihood for success is evident. Once these molecular tools are available, the intent is to investigate the role of glutamine transport in the insulin-dependent diabetic animal to gain clear understanding of the impact that the transporter has on nitrogen metabolism in this and other catabolic states.